Radiation induced bystander effects have been demonstrated with a variety of endpoints, but the mechanism of the phenomenon is not known. While gap junction communication and presence of soluble mediators) are known to play an important role in the bystander response, the precise signaling molecules have yet to be identified. This program project brings together and links 3 projects that all address the common goal of understanding the how and why of the bystander phenomenon. The central hypothesis of the overall program is that the bystander effect involves multiple pathways and that an initiating event in the hit cells and a subsequent downstream signaling step involving the arachidonic acid cascade in the bystander cells play an important role in mediating the process. A number of innovative and inter-related approaches are proposed to address these goals. Project 1 will harness the power of microarray profiling and functional genomics in order to gain insight into the cascade of signaling events between cellular targets and between cells. This study will be extended to a 3D tissue model as well as to single cells. Project 2 will follow up on the preliminary observations that reactive nitrogen species may be involved in the signaling process and that the COX-2 enzyme is consistently elevated in bystander cells. Project 3 will characterize the role and the underlying mechanisms(s) whereby radiation induced bystander effect contributes to genomic instability. In between the projects, we will examine the gene profiling of nuclear versus cytoplasmic irradiation and whether the latter can induce bystander response in a manner similar to nuclear traversals. These studies are entirely dependent on the technology of the Columbia microbeam, which makes it possible to aim a defined number of a-particles (including one) at either the nucleus or cytoplasm of a cell with a precision of a few microns. The unequivocal demonstration of the bystander effect represents a paradigm shift in radiation biology since generations of students had been taught that heritable effects required the direct deposition of radiant energy in DNA. It is now apparent that the target for heritable damage is not only larger than the DNA, but larger than the cell itself.